Borrelia burgdorferi, the infectious agent of Lyme disease, is transmitted to mammals through the bite of infected Ixodes ticks. Our broad objective is to use a genetic approach to elucidate the molecular mechanisms of adaptation and variation in B. burgdorferi and their roles in the infectious cycle. As an initial step toward genetic manipulation of B. burgdorferi we have developed a method of gene inactivation by allelic exchange, using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker. We chose gyrBr because it is the only antibiotic resistance marker for which there are data demonstrating a selectable phenotype in B. burgdorferi. Although we are excited about the potential of this technique, it has several shortcomings. Homologous recombination via flanking sequences yields the desired targeted insertion very infrequently; most (99.6%) coumermycin-resistant colonies are generated by recombination with the chromosomal gyrB gene. A different selectable marker without a borrelial counterpart would circumvent this problem. We are currently investigating the utility of several additional antibiotic resistance genes as selectable markers. These include kanamycin, chloramphenicol, hygromycin, and lincomycin resistance genes. We are using a technique that results in the intergration of a large segment of foreign DNA (including the new marker) by a single cross-over between gyrB genes on the transforming circular plasmid and the borrelial chromosome. Plasmid integrants are then analyzed to detrmine if the second marker is expressed at an adequate level to confer a selectable phenotype. We have taken the same approach to investigate the utility of green fluorescent protein (GFP) as a reporter gene in B. burgdorferi. We have inactivated several plasmid-borne genes that we suspect are important in the transmission of B. burgdorferi. Our goal is to analyze the phenotype of these mutant spirochetes in the infectious cycle. However, all gene inactivations to date have been done with an uncloned, non-infectious variant of strain B31; attempts to transform an infectious clone of strain B31 have been unsuccessful. In collaboration with Drs. Anguita and Fikrig at Yale University we have recently overcome this obstacle by using a clone of a different isolate, N40, that can be transformed by electroporation and is also infectious for mice and transmissible by ticks. We are currently attempting to inactivate the genes of interest in this infectious clone. B. burgdorferi naturally contains a large number of extrachromosomal DNAs (plasmids). Few of the plasmid-borne genes are similar to other bacterial genes, suggesting they provide functions unique to the spirochete. We have undertaken the study of several of these plasmids with the goal of understanding their roles in the life cycle of B. burgdorferi. We have demonstrated that the 32 kb circular plasmid (cp32) is actually a family of at least 7 different plasmids. Extended regions of near identity are interrupted by highly divergent loci. Linkage analysis of divergent loci indicate past recombination events among the cp32s. The cp32 sequences were not included in the recently published B. burgdorferi genomic sequence because their high degree of DNA sequence similarity prevented unambiguous assembly of individual sequences. Collaborative studies with Dr. Casjens at the University of Utah are currently underway to assist in the assembly of these related plasmids.